Re: [Histonet] In situ hybridization on insects: fixing, processing & cuticle softening advice

From:Bruce Webber



... and here is the original email, given that the previous email does
not seem to link up to the original message thread.

On 05/04/2007 16:38, Bruce Webber wrote:
> Hi Histonetters,
>
> I am looking for advice on preparing and processing small (c. 2-3mm
> long) adult ants (i.e. with a well-formed chitinous exoskeleton) for
> in situ hybridization (ISH) detection of bacteria (using DNA probes to
> target RNA).  The ants are transverse sectioned into 3 parts before
> fixing to allow greater penetration into each of the 3 main body cavities.
>
> I've trawled the archives for various options and have consulted my
> histological mentor, Bruce Abaloz, but after not finding what I was
> looking for, I would greatly appreciate advice, opinions and
> recommendations from others.  I do promise to post a summary of any
> responses together with any methodological tips I found along the way!
>
> The main problem I see is dealing with the cuticle to allow for good
> sectioning (5-8um), but using chemicals & methods that don't interfere
> with ISH, damage the target RNA, or interfere with the efficiency of
> the ISH probes.  My understanding is that the following factors need
> to be addressed:
>
> 1) Fixing the tissue: 
> The only available material available at this stage was fixed in 2%
> gluteraldehyde (12h at 4C) and then stored (> 3 weeks) in 80% EtOH
> (meaning that gut excision is not an option due to brittle material). 
> In the future, 4% paraformaldehyde, 10% neutral buffered formalin or
> just straight into 70% EtOH (with slightly shorter storage times)
> could also be considered as options if people feel that they may be
> better (the latter option would certainly increase the availability of
> material).
>
> 2) Softening the cuticle: 
> IMHO this is the biggest potential area for chemical
> damage/interference with ISH.  Proposals I am aware of that would not
> be compatible with ISH due to acidic damage of the RNA are [1]
> Perenyi's fluid (4:3:3, 10% nitric acid:100% EtOH:0.05% chromic
> acid);  [2] Diaphanol (50% glacial acetic acid saturated with ClO2);
> and [3] Bouin's fixative (3:1:2, saturated picric
> acid:formaldehyde:glacial acetic acid).
>
> However, other suggestions from previous posts which I know very
> little about include [4] chloral hydrate processing (after tissue
> dehydration, melt equal parts of chloral hydrate & phenol and immerse
> tissue for up to 7 days, followed by chloroform as an intermediary
> agent);  [5] Mollifex Gurr (glycerol, phenol, acetone and EtOH
> solution, applied to the cutting surface of the paraffin block);  [6]
> Nair (as in the hair removal cream - thioglycolate salts & calcium
> hydroxide, between fixing and processing, soak the tissue for c.
> 24hrs); and [7] Phenol (after fixation, soak fixed specimens in 4%
> phenol (in 80% EtOH) for 24 hours).  Lastly, there is 1% DMSO (added
> to the fixative), but given that the ant is chopped into 3 sections,
> this may not be necessary to ensure adequate penetration (but does
> DMSO also improve sectioning?).
>
> 3) Processing the tissue: 
> The ongoing debate of xylene v histoclear (or histosolve)..... 
> Histoclear is probably safer, but does xylene produce better results? 
> Has anyone had experience with either on similar invertebrate
> material?  Others have suggested using isopropyl alcohol (instead of
> EtOH) for dehydration as is tends to have a less hardening effect on
> the tissue.
>
> 4) Material for embedding: 
> I've seen it argued that tougher material such as that containing
> chitinous exoskeletons benefits from using a harder embedding medium
> (e.g. TissuePrep 2 from Fisher) or one with better infiltration (e.g.
> Paraplast X-tra) over standard paraffin.  I've also read that
> Paraplast Plus (containing DMSO) makes sectioning of cuticles easier. 
> Any opinions/recommendations/experience with similar material and any
> of these embedding mediums would be appreciated!
>
> Thank you all in advance for your help with this matter (I apologise
> for the length of this post!).
>
> Bruce Webber
> Centre d'Ecologie Fonctionnelle et Evolutive, CNRS, France
>
> (Key words: cuticle, exoskeleton, chitin, insect, invertebrate, ant,
> in situ hybridization, ISH, softening.)

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